Journal of Structural Biology - Department Of Biological Sciences Hunter …biology.hunter.cuny.edu/cellbio/Feinstein Cell Bio 2013... · 2011. 11. 30. · californica multiple nucleopolyhedrovirus

Journal of Structural Biology xxx (2011) xxx–xxx

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Journal of Structural Biology

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Nuclear transport of baculovirus: Revealing the nuclear pore complex passage

Shelly Au, Nelly Panté ⇑Department of Zoology, University of British Columbia, 6270 University Boulevard, Vancouver, British Columbia, Canada V6T 1Z4

a r t i c l e i n f o

Article history:Available online xxxx

Keywords:Electron microscopyElectron tomographyNuclear importNuclear pore complexBaculovirus capsid

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Abbreviations: AcMNPV, Autographa californica muEM, electron microscopy; FG, phenylalanine–glycine;salt buffer; MBS, modified Barth’s saline; NE, nuclocalization signal; NPCs, nuclear pore complexes;NTRs, nuclear transport receptors; Nups, nucleoporTEM, transmission electron microscope; 3D, three-dimagglutinin.⇑ Corresponding author. Fax: +1 604 822 2416.

E-mail address: [email protected] (N. Panté).

Please cite this article in press as: Au, S., Pantédoi:10.1016/j.jsb.2011.11.006

a b s t r a c t

Baculoviruses are one of the largest viruses that replicate in the nucleus of their host cells. During aninfection the capsid, containing the DNA viral genome, is released into the cytoplasm and delivers thegenome into the nucleus by a mechanism that is largely unknown. Here, we used capsids of the baculo-virus Autographa californica multiple nucleopolyhedrovirus in combination with electron microscopy anddiscovered this capsid crosses the NPC and enters into the nucleus intact, where it releases its genome. Tobetter illustrate the existence of this capsid through the NPC in its native conformation, we reconstructedthe nuclear import event using electron tomography. In addition, using different experimental condi-tions, we were able to visualize the intact capsid interacting with NPC cytoplasmic filaments, as an initialdocking site, and midway through the NPC. Our data suggests the NPC central channel undergoes large-scale rearrangements to allow translocation of the intact 250-nm long baculovirus capsid. We discuss ourresults in the light of the hypothetical models of NPC function.

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1. Introduction

Cellular compartmentalization is vital, in order for the cell tofunction efficiently. The nucleus, being a membrane-enclosed orga-nelle, contributes significantly to the regulation of numerous cellu-lar processes. For example, by controlling the access of certainmacromolecules to the nucleus, nuclear transport can regulate tran-scription, DNA replication, and the cell cycle. The flow of proteins,RNAs, and RNA–protein complexes (such as ribosomal subunits,messenger ribonucleoproteins (RNPs), and splicesomal RNPs) intoand out of the nucleus at a rate of up to 1500 molecules per secondis achieved by nuclear pore complexes (NPCs) embedded within thenuclear envelope (NE) (Kowalczyk et al., 2011; Ribbeck and Gorlich,2001; Strambio-De-Castillia et al., 2010; Wente and Rout, 2010).Extensive electron microscopy (EM) studies have been carried outto elucidate the structure of the NPC, including studies by Ueli Aebiand colleagues using several EM techniques and atomic forcemicroscopy (Reviewed in Elad et al., 2009; Lim et al., 2008a,b; Macoet al., 2006; Pante, 2007; Rowat et al., 2008). In particular, usingstate-of-the-art cryo-electron tomography Aebi and others have

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ltiple nucleopolyhedrovirus;GV, granulovirus; LSB, low-

lear envelope; NLS, nuclearNPV, nucleopolyhedrovirus;

ins; RNP, ribonucleoprotein;ensional; WGA, wheat germ

, N. Nuclear transport of baculo

led to progressive improvements of the three-dimensional (3D)models of the NPC, the latest with a resolution of 6 nm (Becket al., 2007; Frenkiel-Krispin et al., 2010). According to these studies,the NPC consists of a membrane-embedded scaffold ring andperipheral components that extend into the cytoplasm (eightcytoplasmic filaments) and nucleus (the nuclear basket). TheNE-embedded ring has a diameter of 125 nm, is 70 nm in height,and contains a large central channel of about 50 nm in diameter(Beck et al., 2004, 2007; Frenkiel-Krispin et al., 2010; Stoffler et al.,1999). This central channel acts as a molecular sieve, allowing pas-sive diffusion of ions and molecules smaller than 9 nm in diameter(or proteins smaller than 40 kDa) and selective facilitated transportof larger cargos up to 39 nm in diameter (Pante and Kann, 2002). Theselectivity is dictated by a signal (stretches of amino acids callednuclear localization signals (NLSs) or nuclear export signals) resid-ing on the transported molecule, which is recognized by nucleartransport receptors (NTRs) derived from a family of proteins knownas karyopherins or importins (Chook and Suel, 2011; Strambio-De-Castillia et al., 2010; Terry and Wente, 2009; Wente and Rout,2010).

The main components of the NPC are proteins called nucleopo-rins (Nups). Multiple copies of about 30 different Nups make upthe 120-mDa metazoan NPC (Cronshaw et al., 2002). Nups havebeen classified into three families: integral membrane proteins,which anchor the NPC in the NE; scaffolding Nups, which formthe NPC scaffold structure; and FG-Nups, which are rich in hydro-phobic phenylalanine–glycine (FG) repeat motifs (Brohawn et al.,2009; Tetenbaum-Novatt and Rout, 2010; Walde and Kehlenbach,2010). The FG-repeat regions of these Nups presumably populate

virus: Revealing the nuclear pore complex passage. J. Struct. Biol. (2011),

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2 S. Au, N. Panté / Journal of Structural Biology xxx (2011) xxx–xxx

the central channel of the NPC and form the transport barrier of theNPC. Additionally, FG-Nups have a role in the initial docking of thecargo to the NPC, as interactions of NTRs with peripheral FG-Nupsat the NPC cytoplasmic filaments dock the cargo to the NPC (Grun-wald and Singer, 2010; Hamada et al., 2011; Lowe et al., 2010; Ya-seen and Blobel, 1999).

Despite considerable progress in elucidating the structure andcomposition of the NPC, in identifying nuclear transport signalsand their receptors, and in characterizing the interaction ofFG-Nups and NTRs, the molecular mechanism by which moleculestranslocate through the NPC central channel remains unclear. Sev-eral models have attempted to explain how interaction betweenFG-Nups and NTRs allows for efficient and selective translocationof the cargo through the NPC. Of these models, the Brownian Affin-ity Gate/Virtual Gating model (Lim et al., 2006; Rout et al., 2000,2003) and the Selective Phase model (Frey and Gorlich, 2007; Freyet al., 2006; Ribbeck and Gorlich, 2002) are the best described; theyare also the most debated. A common rationale between these twomodels is that FG-containing Nups form a barrier for nuclearimport, and transport occurs via association and disassociation ofNTRs with the FG repeats. Further studies in this area resulted inalternative models such as the Reduction of Dimensionality model(Peters, 2005) and the more recent Forest model (Yamada et al.,2010).

While biochemical and biophysical measurements and compu-tational simulations have been developed to support each model,these models remain controversial. Direct visualization of largecargos crossing the NPC central channel may be a good strategyto test these hypothetical models. In addition, such studies will fur-ther substantiate the ability of NPCs in setting boundaries for cargoselection, and will provide a clear depiction of the overall structureand dimensions of the NPC central channel when occupied by alarge cargo in transit. Because viral capsids are among the largestcargos that enter the nucleus, studies on the nuclear import of viralcapsids may provide important information that can be used totest the several proposed models for NPC function. At the sametime, these studies will enhance our knowledge about the replica-tion cycle of viruses.

In the past, viruses have been invaluable tools for importantdiscoveries in the field of nuclear transport. For example, the NLSwas first discovered in the large T antigen of the simian virus 40(Kalderon et al., 1984), and the upper size limitation for moleculesto transport through NPC was determined using hepatitis B viruscapsids with diameters of 36 nm (Pante and Kann, 2002). In thisstudy, we used capsids of the insect virus baculovirus Autographacalifornica multiple nucleopolyhedrovirus (AcMNPV) to demon-strate the flexibility of NPCs in nuclear import, and to further dis-sect the life cycle of this virus.

Baculoviruses are large, rod-shaped (30–60 � 250–300 nm),enveloped viruses with a DNA genome that requires the nuclearmachinery for viral replication (Blissard and Rohrmann, 1990).Therefore, the baculovirus genome must enter the host nucleus,but how this is accomplished remains largely unknown. Usingthe Xenopus laevis oocyte cell system, which is transcriptionallyinactive, in combination with EM and electron tomography, weconclude that, prior to disassembly, the baculovirus AcMNPVcapsid vertically interacts with cytoplasmic components of NPCsto gradually enter the nucleus fully intact.

2. Materials and methods

2.1. Virus and oocytes

Recombinant AcMNPV, propagated in Escherichia coli strainDH108 and amplified at a multiplicity of infection of 1 in Spodopterafrugiperda Sf9 cells were kindly provided by Dr. D. Theilmann

Please cite this article in press as: Au, S., Panté, N. Nuclear transport of baculodoi:10.1016/j.jsb.2011.11.006

(Agri-Food Research Centre, Summerland, B.C., Canada). Baculovi-ruses were purified through a continuous 15–60% (w/v) sucrosegradient centrifugation, as described by Shoji et al. (1997) andObregon-Barboza et al. (2007) with the modifications described inAu et al. (2010). To remove the viral envelope, purified virus wastreated with 1% NP40 for 1 h at 30 �C. After this incubation, the sam-ple was washed by centrifugation to remove excess NP40, and dia-lyzed for 24 h as described in Au et al. (2010). The successfulremoval of the viral envelope and the integrity of the capsid wereevaluated by examining the capsid preparation under the EM stain-ing with uranyl acetate, as described in Au et al. (2010).

Stage VI oocytes were surgically removed from narcotized X.laevis, as described by Pante (2006) and Au et al. (2010). Isolatedoocytes were washed with modified Barth’s saline buffer (MBS:88 mM NaCl, 1 mM KCl, 0.82 mM MgSO4, 0.33 mM Ca(NO3)2,0.41 mM CaCl2, 10 mM HEPES, pH 7.5) and treated with collage-nase (5 mg/ml) in calcium-free MBS for 1 h to defolliculate theoocytes.

2.2. Oocyte microinjection

Injection needles were made by heating and pulling micropi-pettes (Microcaps; Drummond) using a micropipette puller(Inject-Matic). Microinjection of Xenopus oocytes was performedas described in Au et al. (2010) using an oocyte microinjector(Inject-Matic). Oocytes were injected with 100 nl of purified capsidsinto the cytoplasm at the white rim separating the animal and veg-etal hemispheres. As controls, oocytes were mock injected with100 nl of TE (10 mM Tris, 1 mM EDTA, pH 8.7).

For time-course experiments, oocytes were microinjected withcapsids, incubated at room temperature in MBS for 2, 4, or 8 h,and prepared for EM as described below. For some experiments, in-jected oocytes were incubated at 4 �C instead of room temperature,in order to trap the capsid at intermediate stages of nuclear import.

A biochemical inhibitor of nuclear transport was also used todepict transport-arrested capsid at the NPC. Oocytes were microin-jected with the lectin wheat germ agglutinin (WGA) conjugatedwith colloidal gold (WGA-gold). Preparation of colloidal gold andconjugation of WGA with the gold particles were performed asdescribed in Pante (2006). As such, 50 nl of WGA-gold was micro-injected into the cytoplasm or 20 nl of WGA-gold was microin-jected into the nucleus of oocytes, and the oocytes wereincubated at room temperature for 2 h. After the incubation period,oocytes were microinjected into their cytoplasm with 100 nl ofcapsids and further incubated at room temperature for 8 h.

2.3. Preparation of injected oocytes for EM

After microinjection and incubation of oocytes for the indicatedtime, oocytes were prepared for embedding and thin-section EMfollowing detailed protocols in Au et al. (2010). Briefly, oocyteswere fixed with 2% glutaraldehyde in MBS overnight at 4 �C. Fixedoocytes were then washed in MBS and the animal hemispheres(which contains the nucleus) were dissected and fixed with 2% glu-taraldehyde in low-salt buffer (LSB: 1 mM KCl, 0.5 mM MgCl2,10 mM HEPES, pH 7.5) for 1 h at room temperature. After this fix-ation, oocytes were washed with LSB, embedded in 2% low-meltingagarose and post-fixed with 1% osmium tetroxide in LSB for 1 h.Fixed oocytes were sequentially dehydrated in increasing concen-trations of ethanol and embedded in Epon 812 (Fluka) as describedby Au et al. (2010).

Thin 50 nm sections through the NE from Epon-embeddedoocytes were cut on a Leica Ultracut Ultramicrotome (Leica Micro-systems) using a diamond knife (Diatome). Sections were collectedon parlodion/carbon-coated copper EM grids, and stained with 2%uranyl acetate for 15 min and 2% lead citrate for 5 min.



S. Au, N. Panté / Journal of Structural Biology xxx (2011) xxx–xxx 3

2.4. Electron microscopy and tomography

Samples were examined using a Hitachi-7600 transmissionelectron microscope (TEM) operated at an acceleration voltage of80 kV. Micrographs were digitally recorded using a 1-megapixelAMT Advantage CCD camera (ORCA; Hamamatsu Photonics).

For electron tomography, 200 nm thick sections through the NEwere made, and sections were placed on slot grids coated with 1%formvar. Single axis tilt series of 200 nm samples were recordedautomatically over tilt angles ranging from �30� to +30� in two de-gree increments, and then from �70� to +70� every degree on a FEITecnai G2 F20 TEM operated at 200 kV. Tomograms were acquiredusing FEI’s TEM tomography software and reconstructed usingFEI’s Inspect3D software.

3. Results

3.1. Purified capsids are imported into Xenopus oocyte nuclei

In order to visualize the nuclear import of baculovirus capsids(referred to as capsids henceforth) by EM, we purified the capsidsfrom Sf9 cells infected with baculovirus and microinjected theseinto the cytoplasm of Xenopus oocytes (a system well suited forthe study of nuclear import because it enables us to visualizewell-preserved NPCs and NPC-arrested cargos). Before microinjec-tion, the purified capsids were examined by EM after staining withuranyl acetate to evaluate the purity and integrity of the capsids.As documented in Fig. 1, the purification of the capsid by our pro-tocol was effective, yielding typical rod-shaped capsids that had a

Fig.1. Electron micrographs of purified baculovirus AcMNPV capsids stained withuranyl acetate. The micrograph in (A) documents that the purified capsids werevariable in length. The micrographs in (B) document the morphology of the capsidwith its two distinct ends; a blunt end and a conical end with a small protuberance(arrows). Scale bars, 200 nm in (A) and 50 nm in (B).


diameter of 30 nm and variable length of 250–300 nm (Fig. 1A). Inthis preparation, the virus was completely devoid of the envelopeand the capsids appeared electron dense, which is an indicationthat they contain the viral genetic material. These capsids hadthe expected morphology with two distinct ends: one end bluntand one end conical with a small protuberance (arrows in Fig. 1B).

We then microinjected these capsids into the cytoplasm ofXenopus oocytes and followed their fate by EM after the injectedoocytes were embedded in Epon and thin sectioned. To allowobservation of the large capsids at the NPCs, oocytes were incu-bated at room temperature at different times. Our time-courseexperiment showed that at 2 h post-microinjection, about a quar-ter of the capsids were in the cytoplasm, away from the NE (Fig. 2Aand E), while the remaining capsids were already at the NPC(Fig. 2B and E). After 4 h, however, almost all of the capsids wereseen docked at the NPC or very close to the NPC (within a distanceof 100 nm from the NPC; Fig. 2C and F). In the micrographs show-ing capsids at the NPC, the capsids were interacting vertically withthe NPC, and in most of the micrographs we were able to distin-guish the conical end of the capsid at the NPC and the blunt endaway from the NPC. Some capsids were also found inside thenucleus after 4 h post-microinjection (Fig. 2D); however, capsidsdevoid of DNA (empty capsids, which are not electron dense) ordisassembled capsids were not observed. Eight hours post-micro-injection was a sufficient amount of time for most capsids to enterthe nucleus, as both the cytoplasm and nucleus were capsid-free(data not shown). This also suggests that by 8 h, capsid disassem-bly has occurred and the DNA genome has been released into thenucleus. Unfortunately, we were unable to detect the order inwhich these events occurred.

3.2. Capsids remain intact while vertically traversing the NPC

Electron micrographs from oocytes that were incubated for3.5 h post-microinjection at room temperature showed the capsidvertically traversing the NPC and some of them midway throughthe NPC (Fig. 3). Remarkably, the NPCs containing capsids in transitappeared less electron-dense than neighbouring NPCs not engagedin capsid nuclear transport. In particular, an area of 8–10 nm indiameter surrounding the capsid appeared completely empty, asif all the material normally filling the NPC central channel hadretracted, presumably to allow movement of the capsid acrossthe NPC.

In order to compensate for the fact that thin sections of 50 nmthrough the NE may not be representative of the NPC as a whole,we obtained thicker sections of 200 nm to generate tomogramsof capsids in the midst of being imported into the nucleus throughthe NPCs. EM tomograms showed that both the capsid and NPCremain intact during this translocation event (Fig. 4 and Supple-mentary video 1). Similarly, an unfilled area around the capsidwhilst traveling through the central channel can be seen inFig. 4. Tomographic reconstruction of this event better illustratedan intact capsid traversing the NPC (Supplementary video 2). Viathe 3D view, we observed intact capsids in transit through theNPCs, documenting that, in fact, the intact capsid crosses theNPC. Likewise, the NPC was seen to wrap around the capsid, furtherdemonstrating the movement of the capsid within the centralchannel of the NPC.

3.3. Targeting of capsids to the NPC is delayed at low temperature

Biochemical and physiological inhibitors of nuclear transporthave been used extensively to arrest imported molecules at inter-mediate stages of its passage into the nucleus. Accumulation ofcargos at the NPC cytoplasmic filaments and at the cytoplasmicentrance of the NPC central channel have been observed by EM



Fig.2. Electron micrographs of Xenopus laevis oocytes that have been microinjected with baculovirus AcMNPV capsids and incubated at room temperature for two (A and B)or 4 h (C and D). Bar graphs (E and F) show the percentage of capsids found associated with the NPC, 100 nm away from the NPC, and in the cytoplasm from experimentsperformed as indicated above. A total of 150 capsids were scored for each condition from three different experiments. Capsids were found in the cytoplasm (A), at the NPC orat 100 nm from the NPC by 2 h post-microinjection (B). Most capsids were docked at the NPC (C) by 4 h post-microinjection, and some were inside the nucleus (D) by thistime. Scale bar, 200 nm. n, nucleus; c, cytoplasm. Arrows point to capsids.

Fig.3. Electron micrographs of NPC cross-sections from Xenopus oocytes that have been microinjected with baculovirus AcMNPV capsid and incubated at room temperaturefor 3.5 h. Capsids of 250–300 nm in length are seen traversing the NPCs. Capsids appear fully intact in its native conformation while crossing the NPC. Note the capsid in themiddle panel appear shorter due to the variability in the length of these capsids, as documented in Fig. 1. Scale bar, 100 nm. n, nucleus; c, cytoplasm.


when nuclear import is inhibited at 4 �C (Pante, 2007; Pante andAebi, 1996; Rollenhagen and Pante, 2006; Rollenhagen et al.,2003). Low temperature inhibits the translocation of cargo through


the NPC but does not inhibit its initial docking at the NPC. It is sur-mised that this condition yielded an increased amount of capsids atthe cytoplasmic face of the NPC; however, we found that in oocytes



Fig.4. Tomographic x-y slices spaced approximately 20 nm through the 3D volume of the tomographic tilt series of a capsid in the midst of being imported into the nucleusthrough the NPCs. Scale bar, 200 nm. n, nucleus; c, cytoplasm. Arrow points to the capsids.


incubated for 2 h at 4 �C post-microinjection, 88% of the capsidsremained in the cytoplasm far from the nucleus (Fig. 5A and C),in contrast to the 23% observed when oocytes were incubated for2 h at room temperature (Fig. 2A and E). Similarly, significantlymore capsids were found docked at the NPC after 4 h incubationat room temperature (Fig. 2D and F) than after 4 h incubation at4 �C (Fig. 5B and D). The delayed progress of capsids transitingtowards the nucleus suggests that metabolic energy is the drivingforce that allows the capsid to move within the cytoplasm. Ourdata is in agreement with the previous findings that actin-basedmotility drives the capsid towards the nucleus (Charlton andVolkman, 1993; Ohkawa and Volkman, 1999; Ohkawa et al., 2010).

3.4. Initial docking of the capsid occurs at the cytoplasmic filaments ofNPCs

The low temperature experiments also demonstrated that theNPC cytoplasmic filaments act as the first binding sites for the cap-sid prior to capsid translocation through the NPC. Oocytes thatwere incubated for 4 h at 4 �C yielded 87% of capsid at the cytoplas-mic face of the NPC at about 100 nm from the center of the NPC,and in most of the micrographs the cytoplasmic filaments wereclearly depicted (Fig. 5B). In most of the micrographs, we were alsoable to visualize the conical end of the capsid at the NPC, and theblunt end of the capsid away from the NPC (see for example,Fig. 5B left panel).

To confirm our results of the initial binding of the capsid to theNPC cytoplasmic filaments under conditions that do not delay thetargeting of the capsid to the NPC, we used WGA, a well character-ized inhibitor of nuclear import that binds O-linked N-acetyl


glucosamine residues on glycosylated Nups, thereby blocking theinteraction between NTRs and Nups and inhibiting nuclear trans-port (Finlay et al., 1987; Newmeyer and Forbes, 1988). For theseexperiments, we conjugated 10 nm gold particles to WGA (to visu-alize the binding of WGA to the NPC) and the WGA-gold complexeswere pre-microinjected into the cytoplasm of the oocytes. After 2 hof incubation at room temperature, the oocytes were again micro-injected into their cytoplasm with capsids and incubated for 8 h atroom temperature. After 8 h, WGA-gold particles had accumulatedat the entrance of the NPC central channel, blocking the transloca-tion of the capsid through the NPC, and the capsids remained at theNPC cytoplasmic face at a distance of about 100 nm from the cen-tre of the NPC (Fig. 6A).

We also attempted to block the NPC from its nuclear side bypre-microinjecting WGA-gold into the nucleus of the oocytes. Sim-ilar to the cytoplasmic injection, nuclear injected oocytes wereincubated for 2 h, post-microinjected with capsids, and furtherincubated for 8 h at room temperature. Under these conditions,the WGA-gold particles were within the NPC central channel, whilethe capsid remained on the cytoplasmic face of the NPC (Fig. 6B).Furthermore, at 8 h post-microinjection we observed capsids inthe cytoplasm when the NPCs were blocked by WGA, an occur-rence that was not observed when NPCs were uninhibited. Ourdata demonstrates that the NPC cytoplasmic filaments are theinitial docking sites for the capsids.

4. Discussion

Despite recent advances in microscopic techniques to help cor-relate the structure of the NPC with its function, the molecular



Fig.5. Electron micrograph of Xenopus laevis oocytes that have been microinjected with baculovirus AcMNPV capsids and incubated at 4 �C for 2 h (A) or 4 h (B). Bar graphs (Cand D) show the percentage of capsids found associated with the NPC, 100 nm away from the NPC, and in the cytoplasm from experiments performed as indicated above. Atotal of 150 capsids were scored for each condition from three different experiments. Most capsids were found in the cytoplasm at 2 h post-microinjection (A), while themajority of capsids were docked at the NPC at 4 h post-microinjection (B). No capsids were found inside the nucleus under these conditions. Scale bar, 200 nm. n, nucleus; c,cytoplasm. Arrows point to capsids.

Fig.6. Electron micrograph of Xenopus oocytes that were microinjected with WGA-gold into either the cytoplasm (A) or nucleus (B), incubated at room temperature for 2 h,followed by cytoplasmic injection of baculovirus AcMNPV capsids and further incubated for 8 h. When NPCs were inhibited by WGA-gold particles, capsids remainedinteracting with the NPC cytoplasmic filaments 8 h post-microinjection. No capsids were found inside the nucleus when NPCs were inhibited with WGA. Scale bar, 200 nm. n,nucleus; c, cytoplasm. Black arrows point to capsids and white arrowheads point to WGA-gold particles at NPCs.


mechanism of translocation through the NPC remains elusive.Although several models have been proposed in recent years toexplain this mechanism, due to the lack of in vivo experimental set-ups to test these models, they remain controversial and are a majortopic of debate. One feasible strategy to test these models focuseson the visualization of large cargos crossing the NPC. We chose tostudy the nuclear import of baculovirus to provide a more conciseunderstanding of both how the NPC functions and the strategyused by this virus to enter the nucleus.

With a diameter of 30 nm, the baculovirus capsid is small enoughto cross the NPC without apparent deformation; however, directdemonstration of the actual translocation of the capsid throughthe NPC has not been reported. Previous attempts to visualize this


using EM yielded contradictory results. The first study reportedcapsids from the baculovirus genus granulovirus (GV) docking atthe NPC of infected cells, but not inside the nucleus (Summers,1971). This suggested a mechanism of DNA nuclear import similarto the herpes simplex virus-1, which attaches to the cytoplasmic sideof the NPC and ejects its nucleic acid into the nucleus through theNPC, leaving empty capsids at the NPC (Sodeik et al., 1997). Otherstudies using the baculovirus genus nucleopolyhedrovirus (NPV)inoculated into larvae observed capsids in both the cytoplasm andthe nucleus (Granados, 1978; Granados and Lawler, 1981; Raghowand Grace, 1974); however, it was unclear whether the observedcapsids entered the nucleus during mitosis when the nuclear mem-brane was absent, or via NPCs. Subsequent studies following the




infection of insect cells in culture reported capsids in both the cyto-plasm and the nucleus of the infected cells as early as 2 h post-infec-tion (Carsten et al., 1979), indicating that the capsid crosses the NPCintact. More recently, experiments with tissue-cultured cells, ar-rested at G1/S phase revealed capsids on the cytoplasmic side ofthe NPC as well as inside the nucleus (van Loo et al., 2001), thus sup-porting a mechanism in which the entire baculovirus capsid crossesthe NPC and genome release occurs in the nucleus. These contradic-tory findings suggest that perhaps the mechanism of nuclear importof the baculovirus capsid could be genus specific. In all these studiesreporting capsids inside the nucleus, however, it was unclearwhether the capsids observed in the nucleus were imported throughthe NPC or were made during the course of infection. To decipher thisdifference and to depict the mode of nuclear entry employed by thebaculovirus capsid, we used a non-replicating and non-dividing cellsystem: X. laevis oocytes.

Upon microinjection of baculovirus capsids into the Xenopusoocyte cytoplasm and analysis of the oocytes by EM, we observedcapsids docking at the cytoplasmic side of the NPC in what appearsto be NPC cytoplasmic filaments (Figs. 2B and C and 5B). Capsid inter-action with the NPC appears to be with the conical end, and not withthe blunt end. In addition, we also observed capsids in the nucleus ofthe injected oocytes (Fig. 2D). As baculoviruses do not replicate inXenopus oocytes, the capsids found inside the nucleus must havebeen imported from the cytoplasm through the NPC. Consistent withthis explanation, we depicted the capsid midway through the NPC(Fig. 3), and demonstrated by electron tomography (Fig. 4, Supple-mentary videos 1 and 2) that the capsid crosses the NPC intact. Thus,our data supports a nuclear entry mode for baculovirus from thegenus NPV that involves translocation of the intact capsid throughthe NPC, similar to a DNA virus of similar diameter, the hepatitis Bvirus capsid (Pante and Kann, 2002; Rabe et al., 2003).

The endocytic route is often necessary for viruses to becomecompetent for nuclear import. For example the acidic environmentof the endosome could trigger exposure of NLSs. Given that wemicroinjected baculovirus capsids into the cytoplasm of Xenopusoocytes, we bypassed the endocytic route. Since we still observednuclear import of the injected capsids, our data suggests that eitherthe NLSs were already exposed on the surface of the capsid, ormodifications of the capsid in the cytoplasm facilitated this expo-sure. However, the putative NLSs on the capsid and the NTRs rec-ognizing these remain largely unknown. Our data furtherconfirms previous suggestions that endocytic conditions are notnecessary in the exposure of NLSs for nuclear import of baculoviruscapsids (Salminen et al., 2005). More recently, baculovirions wereshown to be able to enter and infect Sf9 insect cells through anon-endocytic pathway, further illustrating the idea that putativeNLSs are within the capsid itself (Dong et al., 2010).

The delayed progress of capsids transiting through the cytoplasmtowards NPCs when oocytes were incubated at 4 �C suggests that ac-tive transport within the cytoplasm was also hindered.P78/83 capsid protein of AcMNPV has been shown to associate withactin-like structures in the cytoplasm (Charlton and Volkman,1993). More recently, it was demonstrated that when the Arp2/3complex binding region in P78/83 was mutated, viral motility withinthe cytoplasm, as well as viral gene expression was delayed (Ohkawaet al., 2010). Therefore, incubating oocytes at low temperature couldhave impeded active transport via actin within the cytoplasm.

The NPC cytoplasmic filaments are the initial docking sites forseveral molecules undergoing nuclear import. Using WGA as aninhibitor of nuclear import, by both cytoplasmic and nuclear injec-tion of this inhibitor into Xenopus oocytes, we demonstrated thatcapsids remained at the NPC cytoplasmic filaments when WGA-gold particles impeded transport through the NPC central channel(Fig. 6). Capsids were also observed at the NPC cytoplasmic fila-ments when the oocytes were incubated at 4 �C (Fig. 5B). This


finding is in contrast to studies using 14 nm nucleoplasmin-conju-gated gold (Pante and Aebi, 1996), in which gold particles weredocumented to locate at the central channel of the NPC whenmicroinjected into Xenopus oocytes at 4 �C. This difference maybe due to the size of the cargo, and supports the idea that differentmechanisms of nuclear entry exist and size of the cargo could bethe main determinant. Consistent with this, splicesomal RNPs con-jugated with gold particles were observed at the NPC cytoplasmicfilaments (like the baculovirus capsid) and not at the NPC centralchannel (Rollenhagen and Pante, 2006). This spatial differencecould also be the result of a large cargo, such as the viral capsidor the splicesomal RNPs with discreet sites for NTR binding, asopposed to a single gold particle containing numerous copies ofthe same small protein and multiple sites for NTR binding.

Using microinjection of baculovirus capsids into Xenopusoocytes, in combination with electron tomography, we also docu-mented that the central channel of the NPC is able to accommodatea very long (250–300 nm) cargo, which can remain fully intact in itsnative conformation while traversing the NPC lengthwise. A similarsituation was observed for the NPC translocation of the Balbiani ringgranule, a premessenger RNP complex of very large size synthesizedin the larval salivary glands of Chironomus tentans. These granulesare 50 nm in diameter and consist of an RNP ribbon bent into aring-like structure. As the granule is exported from the nucleusthrough the NPC, the ribbon (25 nm in diameter by 135 nm inlength) straightens out and has been shown to occupy the centralchannel of the NPC (Mehlin and Daneholt, 1993; Mehlin et al.,1992, 1995). Both our data with the baculovirus capsid and the pub-lished results for the Balbiani ring granules clearly illustrate the flex-ibility of the NPC central channel. The data also demonstrates thatthe NPC must undergo a large scale of rearrangement to allow suchlarge cargos to occupy the NPC central channel.

When in transit, the capsid appears to occupy the whole NPCcentral channel (Figs. 3 and 4). We also observed some emptyspace within the central channel immediately adjacent to the cap-sid, unlike the electron dense central channel of neighbouring NPCsthat are capsid-free. Considering the width of the capsid (30 nm)and our measurements of the empty space surrounding the NPC-crossing capsid (about 10 nm from each side of the capsid), theNPC central channel expanded to about 50 nm to allow the capsidto pass through it. This is the same value for the dimension of theNPC central channel that was deduced from 3D reconstructions ofcryo-EM of NPCs (Beck et al., 2004, 2007; Frenkiel-Krispin et al.,2010; Stoffler et al., 2003). Our finding indicates that whatever isnormally filling the NPC central channel must completely retract,leaving the NPC in an open state that allows the capsid to travelacross it. This idea of an open state of the NPC was originally pro-posed in earlier studies that suggested the concept of a centralplug/transporter that resides within the NPC central channel (Akey,1990, 1991; Akey and Radermacher, 1993). These studies hypoth-esized that the transporter remains in a closed position when car-gos dock and further dilates when cargos are in transit through theNPC. The existence of an NPC central plug/transporter was sup-ported by early structural analysis of the NPC that documentedthe presence of a massive particle in the central channel. Becausethe size, shape and position of this particle were highly variable,the central plug/transporter was later proposed to be moleculesin transit (Beck et al., 2004; Elad et al., 2009; Stoffler et al., 2003).

More recent models of NPC function propose that the centralchannel is filled by the FG-repeat domains of Nups. The SelectivePhase model assumes that these domains are cross-linked, forminga hydrogel (Ribbeck and Gorlich, 2002). Although recombinant FGdomains can form a hydrogel in vitro (Frey et al., 2006), it would beof importance to further demonstrate how such a hydrogel couldreform after being significantly disrupted, leaving an empty spaceof 50 nm in diameter by 70 nm in height (the entire dimensions of




the NPC central channel) that allows the passage of the baculoviruscapsid. Nevertheless, this model suggests an inverse relationshipbetween cargo size and the rate of nuclear import (Lieleg and Rib-beck, 2011); thus, in order to test this model it will be valuable tomeasure the rate of nuclear import of the baculovirus capsid usinglive-cell imaging.

In another proposed model for NPC function, the FG-repeatdomains of Nups have been suggested to act as a polymer brushthat sweeps away macromolecules from a large corona surround-ing the entrance of the NPC central channel (Lim et al., 2006,2007a,b). The FG corona acts as a barrier to reject non-bindingmolecules, while molecules capable of binding to the FG coronacause local FG repeats to collapse, thereby allowing the moleculeinto the transport channel (Lim et al., 2006). It is possible to imag-ine that as soon as the conical end of the capsid interacts with thelarge FG corona surrounding the cytoplasmic entrance of the NPC,all the FG motifs in the central channel are swept away, leavingthe central channel empty and ready to be transited by the capsid.To demonstrate that this model is applicable for the NPC translo-cation of the baculovirus capsid, it would be worthwhile toperform immuno-EM of FG-Nups and see their distribution onNPC-containing capsids.

A more recent proposed model, the Forest model, hypothesisesthat the central channel is separated into two zones – large mole-cules move through the inner part of the NPC channel, and smallermolecules travel along the outer part of this channel (Yamadaet al., 2010). This model suggests that FG domains converge atthe centre of the NPC, forming a central plug/transporter structureto allow the movement of larger cargos. The presence of a largestructure residing within the central channel is inconsistent withthe translocation of large cargos, such as baculovirus capsids andthe Balbiani ring granules. If such a central plug/transporter exists,its fate when large cargos cross the NPC central channel remains tobe explained.

In summary, our results indicate that whatever is normallyfilling the NPC central channel must be remarkably flexible, andis able to retract itself into the membrane-embedded scaffold ringof the NPC, leaving the entire central channel open for thepassage of the large baculovirus capsid. Although our data doesnot seem to fit with some of the proposed models for NPC func-tion, we do not discard the possibility that the NPC may use dif-ferent modes of translocation depending on the size of the cargobeing translocated. Thus, some of these models may be valid forcargos smaller than the baculovirus capsid. Nevertheless, thetranslocation of a long cargo, such as the baculovirus capsid,supports a model in which the total length and width of the cen-tral channel is completely emptied to accommodate the intactcapsid.

Acknowledgments

We are grateful to Dr. David Theilmann (Agri-Food ResearchCentre, Summerland, B.C., Canada) for providing the Baculovirusinfected cells and for helpful discussions. We also thank BradfordRoss of the BioImaging Facility at University of British Columbiafor help with electron tomography. This work was supported byGrants from the Canada Foundation for Innovation, the CanadianInstitutes of Health Research, and the Natural Sciences andEngineering Research Council of Canada. N.P. is a Michael SmithFoundation for Health Research Senior Scholar.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at doi:10.1016/j.jsb.2011.11.006.


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